Formulations and process for isolating viable microorganism from positive blood cultures

ABSTRACT

Various embodiments disclosed herein provide for reagents and methods for rapidly isolating viable microbial cells, including  S. pneumoniae , from positive blood culture samples. The resulting microbial pellet can be used for both identification and growth-based methods such as antimicrobial susceptibility testing. The buffers described herein may contain a base solution, non-ionic detergents, thiols, and optionally, ammonium chloride. The disclosed methods provide a process for rapidly isolating and concentrating viable microorganism (s) from PBC samples using only one sample preparation tube and centrifugation while removing cellular debris from the mammalian blood cells that may interfere with identification methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 61/604,732, filed Feb. 29, 2012, thedisclosure of which is hereby incorporated herein by reference.

RELATED APPLICATIONS

This application is related in subject matter to U.S. patent applicationSer. No. 13/647,072, filed Oct. 8, 2012, which claims the benefit of thefiling date of U.S. Provisional Application No. 61/544,407, filed Oct.7, 2011, the disclosures of which are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Sepsis is a serious medical condition caused by an overwhelming responseof the host immune system to infection. It can trigger widespreadinflammation, which can give rise to impaired blood flow. As sepsisprogresses, the body's organs can be starved for oxygen and nutrients,causing permanent damage and eventual failure. Left improperly diagnosedor otherwise untreated, the heart may weaken and septic shock can occur,leading to multiple organ failure and death. Blood cultures are requiredto detect the presence of bacteria or yeast in the blood of sepsispatients. If a microorganism is present, (positive blood culture(“PBC”)) the microorganism(s) must be identified and antibioticsusceptibility determined in order to provide appropriate treatment. ThePBC samples are used to isolate, identify and perform antimicrobialsusceptibility testing (“AST”). The microorganism(s) are oftenidentified by methods such as mass spectrometry, including MALDI-TOF/MSor phenotypic growth-based methods, such as Phoenix™ ID.

In order to identify the microorganism(s), perform phenotypic analysison the microorganism, and perform AST testing, intact, viablemicroorganism(s) need(s) to be isolated from the blood cells and othermaterial in the collected sample. For identification of themicroorganism by mass spectrometry, the microbial sample needs to besufficiently free from substances known to interfere with MALDI-TOF/MSidentification, such as blood cell components, other cellular debris,and salts. In addition, the microbial sample needs to be of sufficientquantity in order to obtain a reliable identification. Phenotypicidentification methods, such as Phoenix™ ID, require intact, viablemicroorganism free from substances that may interfere with the enzymaticsubstrates of the assay. For AST testing, such as Phoenix™ AST, themicrobial sample needs to contain viable, unaltered microorganismcapable of growth in the presence of antibiotic, if resistancemechanisms are present, during performance of the assay. It is importantfor all methods to be of sufficient quantity and purity as carryover ofresidual blood or media components will interfere either directly or byfalsely increasing the concentration (turbidity) of microorganism.

Current techniques for isolating viable microorganism from a PBC sampleinclude sub-culturing the microorganism(s), which can take up to 72hours. This results in the delay of treatment or treatment withinappropriate antibiotics.

Certain strains of microorganisms are particularly difficult to isolatefrom a PBC sample while maintaining viability of the organism, such as,for example, Streptococcus pneumoniae (S. pneumoniae). Part of thisdifficulty is traced to the activation of autolysin by S. pneumoniaewhich causes the microbial cells to “self-destruct”. See “Streptococcuspneumoniae Antigen Test Using Positive Blood Culture Bottles as anAlternative Method To Diagnose Pneumococcal Bacteremia”, Journal ofClinical Microbiology, Vol. 43, No. 5, May 2005, p. 2510-2512. Thecurrent method for isolating microorganisms from septic patients,including, S. pneumoniae, includes inoculating blood culture bottles.Once a positive signal is achieved, a portion of the PBC sample isremoved to perform a gram stain and another portion is used tosub-culture the microorganism. Microbial colonies from the sub-cultureare used to perform downstream testing such as identification byMALDI-TOF/MS, phenotypic identification methods, and AST testing.

Additional techniques for isolating viable microorganism(s) from a PBCsample often utilize liquid separation methods containing lysis bufferswith detergents that lyse the blood cells in the PBC sample. Afterlysis, the lysed blood cells can be removed while the microorganism(s)is/are retained. However, the use of these lysis buffers often result incompromised, damaged, or non-viable microorganism(s) which is/areinsufficient for performing certain growth-based identification methodssuch as AST testing.

One such liquid separation method, the Bruker Sepsityper™ system, allowsfor direct testing of the microorganism from a PBC sample byMALDI-TOF/MS without the need for sub-culturing the microorganism. Thismethod uses Sodium Dodecyl Sulfate (“SDS”) and centrifugation togenerate a pellet of microbes. While the Sepsityper™ method willgenerally support MALDI-TOF/MS testing of the microbial pellet, there isinsufficient viability to support growth-based identification methodsand AST methods, due to the interaction of the harsh detergents on themicrobial cell wall.

Prod'hom et al., “Matrix-assisted Laser Desorption Ionization-Time ofFlight Mass Spectrometry for Direct Bacterial Identification fromPositive Blood Culture Pellets”, Journal of Clinical Microbiology, Vol.48, No. 4, p. 1481-1483 (Feb. 17, 2010). discloses a method for lysingerythrocytes in a PBC sample using ammonium chloride to prepare abacterial pellet for MALDI-TOF/MS analysis. However, these methods areinsufficient in obtaining reliable MALDI-TOF/MS data across a panel ofmicroorganism, including S. pneumoniae. In addition, there is noindication that these methods will maintain sufficient viability of themicroorganism for use in growth-based identification methods and ASTtesting.

Hansson et al., “Microfluidic Blood Sample Preparation for Rapid SepsisDiagnostics”, KTH Engineering Sciences, (2012) suggests the use ofdetergents for lysing blood cells and selectively lysing certain typesof blood cells with ammonium chloride. However, this reference is silentwith regard to specific formulations or methods that would allow for theisolation of viable microorganism from a PBC sample that is free frominterfering substances and would allow for multiple downstream testingfrom one PBC sample, such as both MALDI-TOF/MS identification and ASTtesting.

Both M. Drancourt, “Detection of Microorganisms in Blood Specimens UsingMatrix-assisted Laser Desorption Ionization Time-of-flight MassSpectrometry: A Review, Clinical Microbiology and Infection,16:1620-1625, (2010) and WO 2010/100612 to Nassif et al. describemethods for isolating microorganism from a PBC sample which includeremoving red blood cells from a PBC sample by adding saponin and/orammonium chloride to a portion of the PBC sample, centrifuging themixture, and washing the resulting pellet with water to remove residualblood proteins. However, these methods produce inconsistentidentification of the microorganism at the species level across a panelof microorganisms and/or fail to identify S. pneumoniae. Furthermore,there is no indication that these methods result in a microbial pelletwith sufficient viability to perform growth-based testing methods, suchas AST.

Accordingly, it is desirable to develop reagents and methods thatrapidly separate microorganism(s) from a PBC sample while maintainingthe viability of the microorganism(s), so that analytical growth-basedmethods that require cell viability, such as AST testing, can beperformed. Additionally, it is desirable that the reagents and methodscan be used to isolate viable cells from all types of microorganism,including S. pneumoniae.

BRIEF SUMMARY OF THE INVENTION

Various embodiments of the disclosed invention provide for reagents andmethods for rapidly isolating viable microbial cells from a PBC sampleincluding S. pneumoniae. The resulting microbial pellet obtained usingthe various reagents and methods is sufficiently free from interferingsubstances and can be used for identification methods, such asMALDI-TOF/MS, growth-based identification and AST methods. This enablesrapid results without the need for sub-culturing the microorganisms. Theconcentrated mass of viable microbial cells obtained by the variousembodiments can be used for both the direct inoculation of rapid IDsystems, such as MALDI-TOF/MS, and ID/AST testing (AST) by conventionalor automated systems, such as the BD™ Phoenix™ ID/AST system. Thevarious embodiments may also be applicable to other systems, moleculartesting methods, such as polymerase chain reaction (PCR), and othermethods known to one skilled in the art.

Described herein are various “lyse and wash” buffers that can be usedfor both lysing mammalian blood cells and washing away cellular debrisand other blood components, while maintaining viability of themicroorganism(s). The “lyse and wash” buffers described herein contain abase solution comprising, for example, salts, peptones, and othernutrients to protect the microorganism(s), as well as lytic reagents,such as detergents, thiols, and ammonium chloride to remove cellulardebris from the mammalian blood cells.

The methods for isolating microorganism(s) from PBC samples describedherein utilize the “lyse and wash” buffers to rapidly produce a pelletof viable microorganism that can be used for various downstream testingmethods, for example, MALDI-TOF/MS identification and AST testing. Thedisclosed methods provide a process for rapidly isolating andconcentrating microorganism(s) from PBC samples using one samplepreparation tube, a single lyse step, a single wash step, and as littleas one centrifugation step. These methods are easily adapted toautomated systems and do not require sub-culturing of the microorganismin order to maintain cell viability.

The methods described herein do not utilize substances that interferewith methods for identifying the microorganism(s), for example massspectrometry, or with phenotypic methods such as Phoenix™ ID.Furthermore, the methods described herein permit for the rapid isolationof viable microorganism(s) from a single PBC sample in sufficientquantity for use in multiple down-stream analysis, such as,identification by mass spectrometry, phenotypic or growth-basedidentification assays, and AST testing. Furthermore, the methodsdescribed herein prepare a sample for downstream analysis that willidentify a wide panel of microorganisms, including the most difficult oforganisms to characterize, for example, S. pneumoniae.

In another embodiment, a kit is provided which may include, for example,one or more of the “lyse and wash” buffers described herein forisolating viable microorganism(s) from a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the method of one embodiment described herein forthe isolation of viable microorganism(s) from a PBC sample.

FIG. 2 demonstrates the method of one embodiment described herein forthe isolation of viable S. pneumoniae from a PBC sample.

DETAILED DESCRIPTION

Isolating and concentrating the microorganism(s) from PBC samples usingthe “lyse and wash” buffers and methods described herein maintains theviability of the microbial cells, and consequently their response togrowth-based testing, such as AST testing. The methods described hereinalso reduce the amount of other substances in the sample, such ascellular debris, that may interfere with multiple identification methodssuch as MALDI-TOF/MS and Phoenix™ ID. In one embodiment, the buffersdescribed herein are formulated to keep the concentration of salts at aminimum so as to prevent interference with MALDI-TOF/MS identificationanalysis.

In yet another embodiment, the buffers and methods described herein areused to isolate and perform downstream analysis on gram-positivebacteria, gram-negative bacteria, or yeast. In another embodiment, thegram-positive bacteria include S. pneumoniae as well as other species ofStreptococci.

“Lyse and Wash” Buffer

The “lyse and wash” buffers described herein contain a balance ofreagents that stabilize the microorganism(s) as well as lytic reagentsfor lysing blood cells. In one embodiment the buffer contains a basesolution comprising salts, peptones, and other nutrients for stabilizingthe microorganism(s) as well as non-ionic detergents, thiols, andoptionally, ammonium chloride, for lysing blood cells in the sample.

Without being bound by a particular theory, it is believed that whilethe base solution may help to stabilize the microorganism(s), lyticreagents lyse the blood cells and remove interfering cellular debris.The “lyse and wash” buffers described herein provide for the potentialfor multiple independent lytic mechanisms: aqueous (osmotic lysis),detergents (membrane solubilization), thiols (disruption ofprotein-protein interactions in the membranes), and ammonium chloride(red blood cell membranes are effectively permeable to ammonium chlorideand cell lysis occurs due to the unbalanced osmotic pressure of theircolloid content). The combination of multiple lytic reagents allows foreach of the lytic reagents to be less harsh on the microorganism(s) andcan be used for a shorter duration than if used separately.

The base solution may contain a nutrient broth for microorganism(s), anisotonic buffer, peptones, and/or salts. The nutrient broth can be, forexample, Trypticase™ soy broth. In one exemplary embodiment theconcentration of the nutrient broth in the “lyse and wash” buffer isabout 10 g/L to about 50 g/L. Isotonic buffers are clinical buffersknown by one skilled in the art to be compatible with whole bloodsamples. Such buffers are typically mixtures of sodium phosphate,potassium phosphate, and saline, such as sodium chloride or phosphatebuffered saline. In one exemplary embodiment, the concentration ofisotonic buffer is about 1 g/L to about 85 g/L. In another exemplaryembodiment, the isotonic buffer is a phosphate buffer. In oneembodiment, the peptones include casein peptones and/or soy peptones.Additional constituents of the base solution contemplated include, forexample, sodium pyruvate, yeast extract, sodium citrate, meat peptones,and/or dextrose.

In another embodiment, the “lyse and wash” buffer may contain anon-ionic detergent or mixtures of nonionic detergents. In thisembodiment, at least one non-ionic detergent in the “lyse and wash”buffer is a non-ionic hemolytic detergent that selectively lyses bloodcells but does not lyse the microorganism(s). Exemplary non-ionichemolytic detergents contemplated include Triton™ X-100 and saponin.Other non-ionic detergents may include, for example, the Tween® family,the Brij® family, octyl-b-Glucoside, the Tergitol® family, the CYMAL®family, the MEGA family (including Noctanoyl-N-methylglucamine,N-nonanoyl, N-decanoyl), PLURONIC® family such as F-68, digitonins, andthe CHAP family. In one exemplary embodiment, the concentration ofnon-ionic detergent(s) in the “lyse and wash” buffer is about 0.01 g/Lto about 20 g/L. In another embodiment, the “lyse and wash” buffercontains Triton™ X-100 at a concentration of about 6.7 g/L.

In one embodiment, the “lyse and wash” buffer contains Triton™ X-100.The Triton™ X-100 concentration is selected to preserve the viability ofS. pneumoniae. Exemplary embodiments contemplate concentrations ofTriton™ X-100 of up to about 1 g/L. In another embodiment, the “lyse andwash” buffer contains Triton™ X-100 at a concentration of about 0.335g/L. In still yet another embodiment, the “lyse and wash” buffercontains Triton™ X-100 at a concentration of about 0.1 g/L to about 1g/l, about 0.1 g/L to about 0.9 g/l, about 0.1 g/L to about 0.8 g/l,about 0.1 g/L to about 0.7 g/l, about 0.1 g/L to about 0.6 g/l, about0.1 g/L to about 0.5 g/l, about 0.1 g/L to about 0.4 g/l, about 0.2 g/Lto about 1 g/l, about 0.3 g/L to about 1 g/l, about 0.2 g/L to about 0.9g/l, about 0.2 g/L to about 0.8 g/l, about 0.2 g/L to about 0.7 g/l,about 0.2 g/L to about 0.6 g/l, about 0.2 g/L to about 0.5 g/l, about0.2 g/L to about 0.4 g/l, about 0.3 g/L to about 0.9 g/l, about 0.3 g/Lto about 0.8 g/l, about 0.3 g/L to about 0.7 g/l, about 0.3 g/L to about0.6 g/l, about 0.3 g/L to about 0.5 g/l, or about 0.3 g/L to about 0.4g/l. The “lyse and wash” buffers containing a concentration of Triton™X-100 selected to preserve the viability of S. pneumoniae can also beused to isolate viable microbial cells from an organism other than S.pneumoniae without the need for a different “lyse and wash” buffer forthese additional organisms.

In one embodiment, the “lyse and wash” buffer may optionally includeammonium chloride. In another embodiment, the concentration of ammoniumchloride in the “lyse and wash” buffer is about 0.01 g/L to about 80g/L.

Although the Applicant does not wish to be bound by a particular theory,the addition of ammonium chloride or other lytic components can helpmitigate the effects of the other lytic components of the “lyse andwash” buffer on the viability of the microorganism. When using variouslytic reagents, a sufficient quantity is needed in order to lyse theblood cells in the PBC sample. This often has the unwanted effect ofcausing microbial cell death. Detergents, thiols, and ammonium chlorideemploy different mechanisms of action for lysing blood cells. Whencombining multiple lytic mechanisms in one “lyse and wash” buffer, eachof the different lytic components can potentially be used in a loweramount. This potentiality reduces the negative effect of lysing themicrobial cells but may be sufficient for lysing the blood cells,resulting in more efficient blood cell lysis than when using each of thelytic ingredients alone. Therefore, microbial cell viability can bemaintained while unwanted blood cell components can be eliminated.

In one embodiment the “lyse and wash” buffer contains a base solutioncomprising phosphate buffered saline, yeast extract, sodium citrate,meat peptones, dextrose, sodium pyruvate; thiols comprising sodiumthioglycolate, and L-cysteine HCL; non-ionic detergents comprisingsaponin and Triton™ X-100; and optionally PBS.

In another embodiment the “lyse and wash” buffer contains a basesolution comprising Trypticase™ soy broth, yeast extract, sodiumcitrate, meat peptones, sodium pyruvate; thiols comprising L-cysteineHCL and sodium thioglycolate; and non-ionic detergents comprisingsaponin and Triton™ X-100.

In yet another embodiment the “lyse and wash” buffer contains a basesolution comprising Trypticase™ soy broth; thiols comprising L-cysteineHCL and sodium thioglycolate; and non-ionic detergents comprisingsaponin and Triton™ X-100.

In one embodiment the “lyse and wash” buffer comprises: a base solutioncomprising casein peptone at a concentration of about 8 g/L to about 35g/L, sodium chloride at a concentration of about 2 g/L to about 10 g/L,soy peptone at a concentration of about 1.5 g/L to about 15 g/L, andpotassium phosphate at a concentration of about 0.5 g/L to about 5 g/L;thiols comprising L-cysteine at a concentration of about 0.01 g/L toabout 2.5 g/L and sodium thioglycolate at a concentration of about 0.01g/L to about 2.5 g/L; and non-ionic detergents comprising saponin at aconcentration of about 0.01 g/L to about 10 g/L and Triton™ X-100 at aconcentration about 0.01 g/L to about 20 g/L.

In one embodiment the “lyse and wash” buffer comprises a base solutioncomprising casein peptone at a concentration of about 8 g/L to about 35g/L, sodium chloride at a concentration of about 2 g/L to about 10 g/L,soy peptone at a concentration of about 1.5 g/L to about 15 g/L, andpotassium phosphate at a concentration of about 0.5 g/L to about 5 g/L;thiols comprising L-cysteine at a concentration of about 0.01 g/L toabout 2.5 g/L and sodium thioglycolate at a concentration of about 0.01g/L to about 2.5 g/L; non-ionic detergents comprising saponin at aconcentration of about 0.01 g/L to about 10 g/L and Triton™ X-100 at aconcentration of about 0.01 g/L to about 20 g/L; and ammonium chlorideat a concentration of about 0.01 g/L to about 80 g/L.

In one embodiment, an antifoaming agent can be optionally added to the“lyse and wash” buffer for manufacturing convenience. Preferably, theantifoaming agent has no demonstrable impact on performance of the “lyseand wash” buffer. Antifoaming agents are well known to those skilled inthe art and are not described in detail herein. Selection of anantifoaming agent, and the amount, is largely a matter of design choice,and well within the capabilities of those skilled in the art based onthe “lyse and wash” buffer requirements described herein. In oneembodiment that antifoaming agent in the “lyse and wash” buffer is at aconcentration of 0.1 g/L.

The concentrations of the various constituents of the “lyse and wash”buffers described herein represent the final concentrations of eachconstituent in the “lyse and wash” buffer. Often, a 1:1 volume ratio ofPBC sample is mixed with the “lyse and wash” buffer; however, othervolume ratios are contemplated. Accordingly, the concentrations of eachconstituent in the “lyse and wash” buffer can be adjusted to account forchanges in the volume ratio of “lyse and wash” buffer to PBC sample inorder to achieve a desired final concentration of the constituents ofthe “lyse and wash” buffer when mixed with the PBC sample.

In one embodiment, the “lyse and wash” buffer contains both saponin andTriton™ X-100 but does not contain an antifoaming agent. Without beingbound by theory, the combination of saponin and Triton™ X-100 results inmuch less foaming of the treated PBC samples relative to using saponinas the sole non-ionic detergent in the “lyse and wash” buffer. Thisreduced foaming improves workflow, makes removal of the supernatantafter centrifugation easier, and reduces or eliminates the need forantifoaming reagents (i.e., silicone fluids).

In one embodiment the “lyse and wash” buffer includes acholine-containing solution, for example, those choline-containingsolutions as described in U.S. patent application Ser. No. 13/647,072,the contents of which are hereby incorporated herein by reference intheir entirety. Although Applicant does not wish to be bound by aparticular theory, the addition of a choline-containing solution to the“lyse and wash” buffer may inhibit, prevent, and/or mitigate autolysisof the microorganism, particularly S. pneumoniae, in the presence oflytic components of the “lyse and wash” buffer.

In another embodiment, a kit is provided which may comprise, forexample, one or more of the “lyse and wash” buffers described herein forisolating viable microorganism(s) from a PBC sample.

Methods for Isolating Microorganism(s) from a PBC Sample

The methods for isolating microorganism(a) from a sample suspected ofcontaining at least one microorganism, for example a PBC sample,described herein may utilize the various “lyse and wash” bufferscontemplated to rapidly produce a viable microbial pellet that can beused for various downstream testing methods, such as, identification byMALDI-TOF/MS, growth-based phenotypic assays and AST testing. In oneembodiment, the method includes adding a portion of a PBC sample withthe “lyse and wash” buffer to form a mixture. In one embodiment thevolume ratio of PBC sample to “lyse and wash” buffer is 1:1. The mixtureis incubated for a period of time to lyse the blood cells in the BPCsample.

In one embodiment the “lyse and wash” buffer mixed with the portion ofthe PBC sample comprises at least one non-ionic detergent at aconcentration of about 0.01 g/L to about 20 g/L. In another embodiment,the “lyse and wash” buffer contains Triton™ X-100 at a concentration ofabout 6.7 g/L. In yet another embodiment, the “lyse and wash” buffercontains Triton™ X-100 at a concentration up to about 1 g/L. In yetanother embodiment, the “lyse and wash” buffer contains Triton™ X-100 atabout 0.335 g/L. In still yet another embodiment, the “lyse and wash”buffer contains Triton™ X-100 at a concentration of range of about 0.1g/L to about 1 g/l, about 0.1 g/L to about 0.9 g/l, about 0.1 g/L toabout 0.8 g/l, about 0.1 g/L to about 0.7 g/l, about 0.1 g/L to about0.6 g/l, about 0.1 g/L to about 0.5 g/l, about 0.1 g/L to about 0.4 g/l,about 0.2 g/L to about 1 g/l, about 0.3 g/L to about 1 g/l, about 0.2g/L to about 0.9 g/l, about 0.2 g/L to about 0.8 g/l, about 0.2 g/L toabout 0.7 g/l, about 0.2 g/L to about 0.6 g/l, about 0.2 g/L to about0.5 g/l, about 0.2 g/L to about 0.4 g/l, about 0.3 g/L to about 0.9 g/l,about 0.3 g/L to about 0.8 g/l, about 0.3 g/L to about 0.7 g/l, about0.3 g/L to about 0.6 g/l, about 0.3 g/L to about 0.5 g/l, or about 0.3g/L to about 0.4 g/l.

In one embodiment, after the mixture is prepared from combining aportion of the PBC sample with “lyse and wash” buffer, the mixture isincubated for up to about 5 minutes. In another embodiment the mixtureis inverted several times during incubation to ensure even mixing of thePBC sample and “lyse and was” buffer. After incubation, the mixture iscentrifuged to form a pellet containing viable microorganism(s) and asupernatant containing blood cell debris. The range of speed and time ofcentrifugation may be optimized in order to achieve a readilydiscernible pellet in order to easily remove the supernatant withoutdisturbing the pellet. Various variables will affect the speed and timeof centrifugation including, for example, volume of the mixture, sizeand shape of the sample tube, and type of rotor. In one embodiment themixture is centrifuged at a speed of about 100×g to about 5000×g. Inanother embodiment the mixture is centrifuged for up to and includingabout 10 minutes.

After centrifugation the supernatant is discarded while the pellet iswashed with a second volume of the “lyse and wash” buffer. The “lyse andwash” buffer used for the washing step can be the same or differentformulation than the “lyse and wash” buffer used for lysing the bloodcells in the lysing step. After washing the pellet, the mixture iscentrifuged for a second time. The supernatant, containing blood celldebris, is again discarded while the pellet, containing viablemicroorganism is retained. Trace liquids from the top of the pellet canbe removed. Lastly, the pellet may be resuspended in a solution fordownstream analysis. In one embodiment, the pellet is resuspended in asolution at a concentration of at least about 4 McFarland.

The viable microbial pellet resulting from the various embodimentsdescribed herein can be used to prepare a common sample for variousdownstream testing methods including identification by massspectrometry, for example, MALDI-TOF/MS identification, phenotypicgrowth-based identification, for example, Phoenix™ ID, and AST testing,for example, Phoenix™ AST testing. In addition, the entire method can beperformed in one sample tube without the need for transferring samplebetween multiple tubes. Therefore, the methods described herein arereadily adaptable to automated systems.

Techniques such as higher PBC sample volume, multiple aliquots of PBCsample, multiple spins, etc., described herein can be deployed toincrease the number of microorganism(s) in the starting volume toimprove yield. In addition, these methods provide a rapid samplepreparation method and are easily automated. Furthermore, the methodsand buffers described herein subject the blood cells to lysis, removeinterfering substances from the PBC sample, and provide high yields ofviable microorganism(s). In one embodiment, the yield of viablemicrobial pellet can be increased by increasing the starting volume ofPBC sample and/or by performing the isolation method on several aliquotsfrom one PBC sample and combining the resulting microbial pellets intoone sample.

FIG. 1 illustrates one embodiment of the methods described herein. Aportion of a PBC sample, for example 5 ml, is transferred (100) to asample tube. A volume of “lyse and wash” buffer, for example 5 ml, isadded (105) to the PBC sample and mixed (110), for example for 5minutes. The mixture is centrifuged (115), for example at 2200×g for 10minutes, resulting in pellet A containing viable microorganism(s) andsupernatant A containing blood cell debris. Supernatant A is decantedand discarded (120) while pellet A is retained (125). A second volume of“lyse and wash” buffer, for example 5 ml, is added (130) to pellet A toresuspend the pellet. Resuspended pellet A is mixed (135) to create asecond mixture. The second mixture is centrifuged (140), for example at2200×g for 10 minutes, resulting in pellet B containing viablemicroorganism(s) and supernatant B containing additional blood celldebris not removed by the first centrifugation step (115). Supernatant Bis decanted and discarded (145) while pellet B is retained (150). Atleast a portion of pellet B is resuspended (155) in a liquid, forexample, with 600 μl of sterile deionized water, to achieve a densemicrobial suspension, for example a suspension of approximately 4McFarland. The resulting microbial suspension can be used for variousdownstream testing methods (160) such as, for example, identification byMALDI/TOF-MS and phenotypic growth-based identification, for example,identification by the Phoenix™ system, and growth-based AST methods suchas Phoenix™ AST testing.

In one embodiment of the methods for isolating viable microorganismdescribed herein, the PBC sample is incubated with a choline solutionprior to or simultaneously with the “lyse and wash” buffers as describedin U.S. patent application Ser. No. 13/647,072, the contents of which isincorporated herein by reference in its entirety.

In another embodiment, methods are provided for the isolation of viableS. pneumoniae from a PBC sample. These methods include obtaining a bloodsample suspected of containing at least one microorganism. A portion ofthe blood sample is added to an anaerobic blood culture bottle while asecond portion of the blood sample is added to an aerobic blood culturebottle. The bottles are incubated until a positive signal is obtained.An early indication of the presence of S. pneumoniae in the blood sampleis obtained by combining a portion of a aerobic PBC sample with the“lyse and wash” buffer described herein to form a mixture, incubatingthe mixture for a period of time to lyse the blood cells, andcentrifuging the mixture to form a pellet. A pellet that is green incolor provides an early indication of the presence of S. pneumoniae inthe blood sample. In the alternative an early indication of the presenceof S. pneumoniae in the blood sample is obtained by visually observingthe aerobic positive blood culture bottle for a solution green in color.

After this early indication of S. pneumoniae, the anaerobic PBC samplemay be used to isolate viable S. pneumoniae for downstream analysisusing any of the methods described herein for the isolation of viablemicroorganism. In one embodiment, the method includes adding a portionof an anaerobic PBC sample with the “lyse and wash” buffer describedherein to form a mixture. The mixture is incubated for a period of timeto lyse the blood cells in the BPC sample.

In one embodiment, the methods for isolating viable S. pneumoniaeinclude the use of a “lyse and wash” buffer containing Triton™ X-100.The Triton™ X-100 concentration is selected to preserve the viability ofthe S. pneumoniae. A concentration of up to about 1 g/L is contemplated.In another embodiment, the methods for isolating viable S. pneumoniaeinclude the use of a “lyse and wash” buffer containing Triton™ X-100 ofabout 0.335 g/L.

In one embodiment, after the mixture is prepared from combining aportion of the PBC sample with “lyse and wash” buffer, the mixture isincubated for up to and including about 5 minutes. In another embodimentthe mixture is inverted several times during incubation to ensure evenmixing of the PBC sample and “lyse and was” buffer. After incubation,the mixture is centrifuged to form a pellet containing viablemicroorganism(s) and a supernatant containing blood cell debris.

After centrifugation the supernatant is discarded while the pellet,containing viable S. pneumoniae, is washed with a second volume of the“lyse and wash” buffer. The “lyse and wash” buffer used for the washingstep can be the same or different formulation than the “lyse and wash”buffer used for lysing the blood cells in the lysing step. After washingthe pellet, the mixture is centrifuged for a second time. Thesupernatant, containing blood cell debris, is again discarded while thepellet, containing viable S. pneumoniae, is retained. Lastly, the pelletmay be resuspended in a solution for downstream analysis.

One embodiment of the method for isolating viable S. pneumoniae from aPBC sample is illustrated in FIG. 2. A blood sample that is suspected ofcontaining S. pneumoniae is obtained (200). A portion of the bloodsample (for example 10 ml) is added (205) to an anaerobic blood culturebottle while a second portion of the blood sample (for example 10 ml) isadded (210) to an aerobic blood culture bottle. A positive signal isobtained (215) in both the anaerobic and aerobic PBC bottles indicatingthe presence of a microorganism. A portion of the aerobic PBC sample,for example 5 ml, is transferred (220) to a tube. A volume of the “lyseand wash” buffer described herein containing a concentration of Triton™X-100 selected to preserve the viability of S. pneumoniae, for example0.335 g/L Triton™ X-100, for example 5 ml, is added (225) to the PBCsample to form a mixture. The mixture is incubated (230) for a period oftime (for example 5 minutes) to lyse the blood cells. The mixture isthen centrifuged (235) to form a pellet green in color which provides anearly indication (240) of the presence of S. pneumoniae in the bloodsample. After an early indication of S. pneumoniae (240) a portion ofthe anaerobic PBC sample is prepared (245) for downstream analysis usingany of the methods described herein for isolating viable microorganismfrom a PBC sample, including, for example, the methods described in FIG.1 using a “lyse and wash” buffer containing a concentration of Triton™X-100 selected to preserve the viability of S. pneumoniae, for example0.335 g/L of Triton™ X-100.

EXAMPLES Example 1

Positive blood culture samples were obtained by inoculating a bloodculture bottle with Enterobacter aerogenes (ATCC 13048) orStaphylococcus aureus (ATCC 43300 MRSA) and incubating until a positivesignal was indicated. A portion to the PBC sample (5 ml) was directlywithdrawn from the bottle and placed into a 15 ml conical tube. Cholinechloride (0.5 ml of 20% choline chloride solution) was added to the PBCsample, incubated at room temperature for 20 minutes, and centrifuged at132×g for 5 minutes in a Beckman J6-MI centrifuge with a JS-4.2 rotor.The supernatant was transferred to a second 15 ml conical tube and mixedwith 5 ml of a “lyse and wash” buffer containing 16 g/L ammoniumchloride 6.7 g/L Triton™ X-100, and BD Bactec Lytic 10 (catalog number442265). The mixture was incubated for 5 minutes at room temperature.The lysed sample was then centrifuged at 1855×g for 10 minutes. Afterremoving the supernatant, the remaining bacteria pellet was resuspendedin 4.5 ml BD Phoenix™ ID broth (Catalog number 246001). A portion of therecovered microorganism was used to determine viability of the organismby plate count. The results of the viability testing by plate countindicated 1.85×10⁹ cfu/ml of E. aerogenes after lysis compared to1.65×10⁹ cfu/ml for the PBC sample prior to lysis. Viability testingalso demonstrated 1.25×10⁸ cfu/ml for S. aureus after lysis compared to2.00×10⁸ cfu/ml for the PBC sample prior to lysis.

A second portion of the recovered bacterial suspension was then furtherdiluted with the BD Phoenix™ ID broth to a concentration of 0.5McFarland and used for Phoenix™ ID/AST testing. The BD Phoenix™ ID/ASTSystem is described in, e.g., U.S. Pat. Nos. 5,922,593, 6,096,272,6,372,485, 6,849,422, and 7,115,384, the contents of which are herebyincorporated herein by reference. A portion of the inoculum preparedabove was used for phenotypic identification by Phoenix™ ID and waspoured into the identification portion of a BD Phoenix™ ID/AST panel(Becton, Dickinson and Company) and sealed with a plastic closure. Asecond portion of the inoculum prepared above was used for AST testingby Phoenix™ AST and was poured into the AST portion of a BD Phoenix™ID/AST panel (Becton, Dickinson and Company) and sealed with a plasticclosure. The antimicrobial susceptibility minimal inhibitoryconcentration (MIC) for a series of antibiotics was calculated for eachof the isolated microbial pellets. The MIC results from the microbialpellets were compared to the MIC results from the same strains obtainedfrom plated pure cultures (“Control”). This comparison indicates whetherthe Test Method provides substantially equivalent results to the ControlMethod, i.e., the rate of essential agreement (EA) between the TestMethod and the Control Method. A dilution difference of 0 indicatesexact agreement between the Test Method and the Control Method. Adilution difference of −1 or +1 indicates that the Test Method isconsidered within EA to the Control Method, i.e., within normal testvariation. Dilution differences outside this range, such as for example−4, +4, −3, +3, −2 and +2, indicates the results are not withinessential agreement for the antibiotic/microorganism tested. The methodsand verification of AST results are further described in “Verificationand Validation of Procedures in the Clinical Microbiology Laboratory”,Cumitech 31, (February 1997, ASM Press).

Recovered bacteria from both PBC samples resulted in a correct Phoenix™identification as E. aerogenes and S. aureus with both samples having a99% confidence value. The Phoenix™ AST results showed 100% essentialagreement (EA) when compared with those samples prepared from platedcolonies, i.e., the dilution difference was within −1 to +1 dilutiondifference. The S. aureus AST results included correct identification ofthe four (4) resistance markers expected for this MRSA strain, includingmethicillin resistent staphylococcus (MRS), mecA-mediated resistentstaphylococcus (mecA), beta-lactamase-producing staphylococcus (BLACT),and staphylococcus MLSb phenotype (STAMLS).

These results demonstrate that the “lyse and wash” buffers describedherein can be used to isolate viable microorganism from a PBC sample fordownstream identification and AST testing.

Example 2

Four positive blood culture samples were obtained by inoculating a bloodculture bottle with either Enterococcus faecium (ATTC 700221),Enterococcus faecalis (ATCC 51299), Staphylococcus aureus (ATCC 43300),or Proteus mirabilis (ATCC 29906). Four different “lyse and wash”buffers were prepared comprising the formulations summarized in Table 1below.

TABLE 1 Buffer #1 Bactec Lytic 10 media and 6.7 g/L Triton ™ X-100Buffer #2 Bactec Lytic 10 media, 3.35 g/L Triton ™ X-100, and 16 g/Lammonium chloride Buffer #3 Bactec Lytic 10 media, 3.35 g/L Triton ™X-100, and 40 g/L ammonium chloride Buffer #4 Bactec Lytic 10 media,3.35 g/L Triton ™ X-100, and 80 g/L ammonium chloride

Each of the four “lyse and wash” buffers were used to prepare amicrobial pellet from each of the four PBC samples for a total of 16samples prepared. To isolate viable microorganism, a portion of each PBCsample (5 ml) was pre-treated with 0.5 ml of a 20% choline chloridesolution for 20 minutes in a 15 ml conical tube. After incubation withthe choline chloride solution, 8 ml of each “lyse and wash” buffer wasadded to the sample and mixed for 10 minutes. The sample was thencentrifuged at 1855×g for 10 minutes. The supernatant was decanted anddiscarded while the pellet was washed by resuspending the pellet in thesame tube with 5 ml 3.4 g/L Triton™ X-100 in BD Bactec Lytic 10 media(catalog number 442265) without ammonium chloride. The resuspendedpellet was allowed to mix for 5 minutes followed by centrifugation at3000 rpm 1855×g for 10 minutes. The supernatant was removed anddiscarded while the pellet was used for both MALDI-TOF/MS identificationand Phoenix™ ID/AST testing as described below.

A portion of the bacterial pellet prepared above was resuspended in amicrocentrifuge tube containing 0.6 ml sterile deionized water. Aportion of the suspension (1 μl) was spotted onto a MALDI-TOF/MS targetplate for bacterial identification analysis and allowed to air dry.Formic acid (1-2 μl of a 70% aqueous solution) was overlaid onto thedried, spotted sample and allowed to air dry. The dried sample wasoverlaid with 1 μl of MALDI-TOF/MS matrix solution (MALDI-MS matrixsolution prepared by dissolving 2.5 mg of HCCA in 250 μl of 2.5%trifluoroacetic acid and 47.5% acetonitrile in de-ionized water) andallowed to air dry before identification by MALDI-TOF mass spectrometry.All mass spectrometry data was recorded on a Bruker Microflex™ LT withBiotyper 2.0 software. The methods of preparing the isolated microbialpellet and identifying the microorganism by the mass spectrometry arefurther described in U.S. application Ser. No. 13/636,944, filed Aug.31, 2012, which is hereby incorporated herein by reference. AMALDI-TOF/MS score greater than or equal to 2.0 indicates identificationto the species level. A MALDI-TOF/MS score between 1.7 and 1.999indicates identification to the genus level. And a MALDI-TOF/MS scoreless than 1.7 indicates no identification or a non-reliableidentification. The results are summarized in Table 2 below. A “yes”indicates that a correct identification was obtained at least to thegenus level, i.e., with a MALDI score of at least 1.7.

A second portion of the suspension prepared for MALDI-TOF/MS asdescribed above was further diluted with Phoenix™ ID broth toapproximately 0.5 McFarland and used to inoculate a Phoenix™ AST panelas described in Example 1. The results are summarized in Table 2 below.For the Phoenix™ AST results, a “yes” represents those samples that werein essential agreement between the control sample (plated colonies),i.e., those samples with a dilution difference of only +1 or −1.

TABLE 2 MALDI-TOF/MS Identification Phoenix ™ AST Buffer #1 Yes Yes E.faecium Buffer #1 Yes Yes E. faecalis Buffer #1 Yes Yes S. aureus Buffer#1 Yes Yes P. mirabilis Buffer #2 Yes Yes E. faecium Buffer #2 Yes YesE. faecalis Buffer #2 Yes Yes S. aureus Buffer #2 Yes Yes P. mirabilisBuffer #3 Yes Yes E. faecium Buffer #3 Yes Yes E. faecalis Buffer #3 YesYes S. aureus Buffer #3 Yes Yes P. mirabilis Buffer #4 Yes Yes E.faecium Buffer #4 Yes Yes E. faecalis Buffer #4 Yes Yes S. aureus Buffer#4 No Yes P. mirabilis Total 15/16 16/16

The results demonstrate that various “lyse and wash” buffers can be usedto isolate viable microorganism from a PBC sample for downstreamidentification by mass spectrometry and AST testing across a panel ofmicroorganisms.

Example 3

Three positive blood culture samples were obtained by inoculating ablood culture bottle with either Enterococcus faecalis (VRE ATCC 51299),Klebsiella pneumoniae (ATCC 700603), or Staphylococcus aureus (ATCC43300). Two different “lyse and wash” buffers were prepared comprisingthe formulations summarized in Table 3 below. The only differencebetween buffer #1 and buffer #2 was the addition of 16 g/L ammoniumchloride in buffer #2.

TABLE 3 Buffer #1 27.5 g/L Trypticase ™ Soy Broth, 0.7 g/L L-CysteineHCl, 0.3 g/L Sodium Thioglycolate, 2.6 g/L Saponin, and 6.7 g/L Triton ™X-100. Buffer #2 27.5 g/L Trypticase ™ Soy Broth, 0.7 g/L L-CysteineHCl, 0.3 g/L Sodium Thioglycolate, 2.6 g/L Saponin, 6.7 g/L Triton ™X-100, and 16 g/L ammonium chloride.

Each of the “lyse and wash” buffers of Table 3 were used to prepare amicrobial pellet from each of the three PBC samples for a total of sixsamples prepared. A portion of each PBC sample (5 ml) was mixed with 5ml of each “lyse and wash” buffer. The samples were then centrifuged at2200×g for 10 minutes. The supernatant was decanted and discarded whilethe pellet was washed by resuspending the pellet in the same tube withthe same “lyse and wash” buffer. The resuspended pellet was centrifugedat 2200×g for 10 minutes. The supernatant was removed and discardedwhile the pellet was used for both MALDI-TOF/MS identification andPhoenix™ ID/AST testing as described below.

A portion of the bacterial pellet was resuspended in a microcentrifugetube containing 0.6 ml sterile water. A portion of the suspension (1 μl)was spotted onto a MALDI-TOF/MS target plate for bacteria identificationanalysis as described in Example 2. The results are summarized in Table4 below and indicate whether a correct identification was obtained atleast to the genus level, i.e., with a MALDI score of at least 1.7. Asecond portion of the suspension prepared for MALDI-TOF/MS above wasfurther diluted with Phoenix™ ID broth to approximately 0.5 McFarlandand used to inoculate a Phoenix™ ID panel as described in Example 1. Theresults are summarized in Table 4 below.

TABLE 4 MALDI-TOF/MS Phoenix ™ Identification Identification Buffer #1Yes Yes E. faecalis Buffer #1 Yes Yes K. pneumoniae Buffer #1 Yes Yes S.aureus Buffer #2 Yes Yes E. faecalis Buffer #2 Yes Yes K. pneumoniaeBuffer #2 Yes Yes S. aureus Total 6/6 6/6

The results demonstrate that various “lyse and wash” buffers, with orwithout ammonium chloride, can be used to isolate viable microorganismfrom a PBC sample for downstream identification across a panel ofmicroorganisms.

Example 4

Tests were performed to determine the viability of several differentstrains of S. pneumoniae upon exposure to various “lyse and wash”buffers described herein. Additional tests were performed to determinewhether processing a PBC sample with these various “lyse and wash”buffers sufficiently removes substances that interfere withidentification of the microorganism.

Cell Viability

Three different “lyse and wash” buffers were prepared as summarized inTable 5 below, each with a different amount of Triton™ X-100, i.e., 6.7g/L, 0 g/L, or 0.335 g/L.

TABLE 5 Buffer #1 27.5 g/L Trypticase ™ soy broth, (high Triton ™ X- 0.7g/L L-cysteine HCL, 100) 0.3 g/L sodium thioglycolate, 2.6 g/l saponin,6.7 g/L Triton ™ X-100. Buffer #2 27.5 g/L Trypticase ™ soy broth (noTriton ™ X-100) with glucose, 0.7 g/L L-cysteine HCL, 0.3 g/L sodiumthioglycolate, 2.6 g/l saponin Buffer #3 27.5 g/L Trypticase ™ soy broth(low Triton ™ X-100) with glucose, 0.7 g/L L-cysteine HCL, 0.3 g/Lsodium thioglycolate, 2.6 g/l saponin, and 0.335 g/L Triton ™ X-100.

A volume of each of these buffers (10 ml) was inoculated with 1×10³cfu/ml of either S. pneumoniae non-mucoidal strain (POS3092) or S.pneumoniae mucoidal strain (POS650) and incubated for up to four hours.As a positive control, a 10 ml volume of anaerobic nutrient broth wasalso inoculated with each of the S. pneumoniae strains, as themicroorganisms would be expected to grow in this nutrient broth. Afterinoculation at the time points of 0 hour, 1 hour, 2 hour, and 4 hour, a100 μl volume of the mixture was plated onto agar plates containing 5%sheep blood at a density of approximately 1×10² cfu/plate. The plateswere incubated overnight at 35° C./5% CO₂ at which time the plates weremanually counted for bacterial colonies. The results of the manual platecount are summarized in Table 6 below.

TABLE 6 Buffer 0 hour 1 hour 2 hour 4 hour S. pneumoniae #1 (high) 23 00 0 Non-mucoidal #2 (no) 82 105 125 101 (POS3092) #3 (low) 105 100 70 31Anaerobic 149 130 98 113 Broth (Control) S. pneumoniae #1 (high) 10 0 00 mucoidal #2 (no) 28 37 19 17 (POS650) #3 (low) 36 38 33 28 Anaerobic70 67 74 57 Broth (Control)

As Table 6 demonstrates, viable S. pneumoniae were not obtainedfollowing a 1 hour exposure to a high concentration of Triton™ X-100(buffer #1). However, comparable amounts of viable S. pneumoniae wereobtained using the no Triton™ X-100 (#2) and low Triton™ X-100 (#3)buffers.

Identification by MALDI-TOF/MS

The various “lyse and wash” buffers of Table 5 were used to prepare abacterial pellet from a PBC sample to be used for identification byMALDI-TOF/MS to determine whether the various “lyse and wash” bufferssufficiently cleaned the PBC sample from interfering cellular debris.PBC samples were prepared with five different strains of S. pneumoniaeincluding non-mucoidal strain POS3092, non-mucoidal strain POS3996,non-mucoidal strain POS3999, mucoidal strain POS650, and mucoidal strainPOS532 by inoculating both an aerobic and anaerobic bottle for each ofthe strains. The microorganism was isolated from each of the PBC samplesusing each of the “lyse and wash” buffers of Table 5 by adding to eachPBC sample a volume of the lyse and wash” buffer at a ratio of 1:1. Themixtures were mixed on a Nutator™ for 5 minutes. The mixture wascentrifuged at 2200×g for 10 minutes followed by decanting anddiscarding of the supernatant. To wash the pellet of residual blood celldebris, an additional 5 ml of the same “lyse and wash” buffer was addedto the tube and vortexed to resuspend the pellet. The mixture wascentrifuged at 2200×g for 10 minutes. The supernatant was decanted andexcess liquid removed from the resulting microbial pellet using a cottonapplicator swab. The microbial pellet was resuspended in 600 μl steriledeionized water to achieve an inoculum of microbial suspension ofapproximately 4 McFarland.

The resulting pellet was prepared for identification by MALDI-TOF/MS bythe methods described in Example 2 above. The MALDI-TOF/MS scores aresummarized in Table 7 below.

TABLE 7 S. pneumoniae S. pneumoniae S. pneumoniae Non- Non- Non- S.pneumoniae S. pneumoniae mucoidal mucoidal mucoidal mucoidal mucoidalBuffer (POS3092) (POS3996) (POS3999) (POS650) (POS532) Aerobic 1.651.859 1.558 1.427 1.57 Bottle/ 1.71 1.647 1.442 1.516 1.371 Buffer #11.633 1.814 1.587 1.281 1.372 (high) Anaerobic 1.982 2.179 2.248 1.6981.682 Bottle/ 0 2.251 2.143 0 1.782 Buffer #2 1.479 2.185 2.02 1.7991.779 (no) Aerobic 1.899 1.204 1.817 1.44 1.408 Bottle/ 1.594 1.3381.837 1.377 1.345 Buffer #2 1.729 1.524 1.769 1.269 1.313 (no) Anaerobic1.983 2.286 2.453 2.213 1.972 Bottle/ 1.891 2.309 2.441 2.066 2.085Buffer #3 1.856 2.202 2.458 2.1 2.205 (low) Aerobic 1.952 2.276 2.1141.428 1.206 Bottle/ 2.002 1.971 2.253 1.415 1.375 Buffer #3 2.092 2.1012.18 1.329 1.389 (low)

The results indicate that preparation of a viable microbial pellet froman anaerobic PBC bottle produced significantly better MALDI scores forvarious strains of S. pneumoniae compared to the pellets prepared fromthe aerobic PBC bottle. In addition, the “lyse and wash” buffersdescribed herein containing low amounts of Triton™ X-100 producesignificantly better MALDI scores across the entire panel of S.pneumoniae strains compared to samples prepared with a highconcentration of Triton™ X-100.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. A buffer for isolating and concentrating viable microorganism from apositive blood culture sample comprising a base solution; at least onenon-ionic detergent; at least one thiol; and optionally, ammoniumchloride, wherein the relative amounts of the base solution, the atleast one non-ionic detergent, and the at least one thiol in the bufferare selected to preserve the viability of S. pneumoniae.
 2. The bufferof claim 1, wherein the at least one non-ionic detergent comprisestriton X-100 at a concentration in the buffer of up to about 1 g/L. 3.The buffer of claim 1, wherein the at least one non-ionic detergentcomprises triton X-100 at a concentration in the buffer of about 0.335g/L.
 4. The buffer of claim 1, wherein the base solution comprises atleast one selected from the group consisting of a nutrient broth, anisotonic buffer, a peptone, and a salt.
 5. The buffer of claim 4,wherein concentration of the nutrient broth in the buffer is about 10g/L to about 50 g/L.
 6. The buffer of claim 4, wherein the nutrientbroth comprises trypticase soy broth.
 7. The buffer of claim 4, whereinthe isotonic buffer comprises at least one selected from the groupconsisting of sodium phosphate, potassium phosphate, phosphate bufferedsaline, and saline.
 8. The buffer of claim 4, wherein the concentrationof isotonic buffer in the buffer is about 1 g/L to about 20 g/L.
 9. Thebuffer of claim 4, wherein the peptone is selected from the groupconsisting of casein peptone and soy peptone.
 10. The buffer of claim 4,wherein the base solution further comprises at least one selected fromthe group consisting of sodium pyruvate, yeast extract, sodium citrate,meat peptones, dextrose, and phosphate buffered saline.
 11. The bufferof claim 1, wherein the at least one non-ionic detergent is selectedfrom the group consisting of the Triton family of detergents, saponin,the Brij family of detergents, octyl-b-Glucoside, the Tergitol family ofdetergents, the CYMAL family of detergents, the MEGA family ofdetergents, the PLURONIC family of detergents and the CHAP family ofdetergents.
 12. The buffer of claim 2, further comprising at least oneadditional non-ionic detergent at a concentration in the buffer of about0.01 g/L to about 20 g/L.
 13. The buffer of claim 1, wherein the atleast one thiol is selected from the group consisting of L-cysteine HCL,sodium thioglycolate, mercaptoethylamine, mercaptosuccinic acid,mercaptoethanol, mercaptoethane sulfonic acid, and thioglycerol.
 14. Thebuffer of claim 1, wherein the concentration of the at least one thiolin the buffer is about 0.005 g/L to 4 g/L.
 15. The buffer of claim 1further comprising ammonium chloride.
 16. The buffer of claim 15,wherein the concentration of ammonium chloride in the buffer is about0.01 g/L to about 80 g/L.
 17. The buffer of claim 1 wherein the basesolution comprises phosphate buffered saline, yeast extract, sodiumcitrate, meat peptones, dextrose, and sodium pyruvate; the at least onethiol comprises L-cysteine HCL, and sodium thioglycolate; and the atleast one non-ionic detergent comprises saponin and triton X-100. 18.The buffer of claim 1, wherein the base solution comprises trypticasesoy broth, yeast extract, sodium citrate, meat peptones, and sodiumpyruvate; the at least one thiol comprises L-cysteine HCL and sodiumthioglycolate; and the at least one non-ionic detergent comprisessaponin and triton X-100.
 19. The buffer of claim 1, wherein the basesolution comprises trypticase soy broth; the at least one thiolcomprises L-cysteine HCL and sodium thioglycolate; and the at least onenon-ionic detergent comprises saponin and triton X-100.
 20. The bufferof claim 1, wherein the base solution comprises casein peptone at aconcentration in the buffer of about 8 g/L to about 35 g/L, sodiumchloride at a concentration in the buffer of about 2 g/L to about 10g/L, soy peptone at a concentration in the buffer of about 1.5 g/L toabout 15 g/L, and potassium phosphate at a concentration in the bufferof about 0.5 g/L to about 5 g/L; the at least one thiol comprisesL-cysteine at a concentration in the buffer of about 0.01 g/L to about2.5 g/L and sodium thioglycolate at a concentration in the buffer ofabout 0.01 g/L to about 2.5 g/L; the at least one non-ionic detergentcomprises saponin at a concentration in the buffer of about 0.01 g/L toabout 10 g/L and triton X-100 at a concentration in the buffer of about0.01 g/L to about 20 g/L; and the optional ammonium chloride, ifpresent, is at a concentration in the buffer of about 0.01 g/L to about80 g/L.
 21. The buffer of claim 1, further comprising an antifoamingagent.
 22. The buffer of claim 1, wherein the at least one non-ionicdetergent is a mixture of saponin and triton X-100, and wherein thebuffer does not contain an antifoaming agent.
 23. A method for isolatingand concentrating viable microorganism from a positive blood culturesample comprising: mixing a portion of a positive blood culture samplewith the buffer of claim 1 to produce a mixture, wherein the amount ofthe buffer relative to positive blood culture sample is such that thecells are lysed and the viability of the microorganism in the positiveblood culture sample is preserved; optionally, incubating the mixture;centrifuging the mixture to produce a pellet and a supernatant;discarding the supernatant while retaining the pellet; resuspending thepellet with the buffer to create a resuspended pellet; centrifuging theresuspended pellet to produce a second supernatant and a second pellet;discarding the second supernatant while retaining the second pelletcontaining viable microorganism.
 24. The method of claim 23, wherein theportion of the positive blood culture sample is mixed with an equalvolume of buffer to produce the mixture.
 25. The method of claim 23,further comprising resuspending the second pellet in a solution andperforming at least one downstream testing of the resuspended secondpellet selected from the group consisting of identification of themicroorganism by mass spectrometry, phenotypic identification,antimicrobial susceptibility testing, and molecular testing.
 26. Themethod of claim 23, wherein the microorganism is selected from the groupconsisting of gram-positive bacteria, gram-negative bacteria, and yeast.27. The method of claim 26, wherein the gram-positive bacteria isStreptococcus pneumoniae.
 28. A method for isolating and concentratingviable Streptococcus pneumoniae from a positive blood culture samplecomprising: obtaining a blood sample suspected of containing at leastone microorganism; adding a portion of the blood sample to an anaerobicblood culture bottle while adding a second portion of the blood sampleto an aerobic blood culture bottle; obtaining a positive signal in boththe anaerobic and aerobic positive blood culture bottles; obtaining anearly indication of the presence of Streptococcus pneumoniae in theblood sample by combining a portion of the aerobic positive bloodculture sample with the buffer of claim 1 to form a mixture, incubatingthe mixture to lyse the blood cells in the blood sample, andcentrifuging the mixture to form a pellet green in color which providesan early indication of the presence of Streptococcus pneumoniae in theblood sample; preparing a portion of the anaerobic positive bloodculture sample for downstream analysis comprising: mixing a portion ofthe anaerobic positive blood culture sample with the buffer comprisingtriton X-100 at up to about 1 g/L to produce a mixture; optionally,incubating the mixture; centrifuging the mixture to produce a pellet anda supernatant; discarding the supernatant while retaining the pellet;resuspending the pellet with the buffer to create a resuspended pellet;centrifuging the resuspended pellet to produce a second supernatant anda second pellet; discarding the second supernatant while retaining thesecond pellet containing viable Streptococcus pneumoniae.
 29. The methodof claim 28, wherein the portion of the anaerobic positive blood culturesample is mixed with an equal volume of buffer to produce the mixture.30. The method of claim 28, wherein the portion of the anaerobicpositive blood culture sample is mixed with the buffer comprising aconcentration of triton X-100 in the buffer of about 0.335 g/L toproduce the mixture.
 31. The method of claim 28, further comprisingresuspending the second pellet in a solution and performing at least onedownstream testing of the resuspended second pellet selected from thegroup consisting of identification of the microorganism by massspectrometry, phenotypic identification, antimicrobial susceptibilitytesting, and molecular testing.
 32. A kit for isolating andconcentrating microorganism from a positive blood culture samplecomprising at least one formulation of the buffer of claim 1.